Transcription regulates many cellular processes including cell proliferation and differentiation. We propose to analyze in detail the anatomy of eukaryotic regulatory networks and how they evolve using the budding yeast, Saccharomyces cerevisiae as a model system. An integrative approach will be used to globally analyze the transcriptional circuitry. The role of each S. cerevisiae transcription factor will be elucidated by comparing gene expression patterns in mutant strains lacking transcription factors with those of wild type cells. The results of these experiments will be combined with binding site information with chromatin immunoprecipitation experiments thus providing a direct indication of the role of each factor in regulation of gene expression. To determine which factors function together and explore the combinatorial code of gene regulation, factors that bind upstream of common gene targets will be identified and tested for interactions in vitro and in vivo. Finally, the divergence of regulatory networks will be examined by comparing the transcriptional regulation of the filamentous network in 5. cerevisiae and related yeasts, including the pathogen Candida albicans. The divergence of transcriptional control will be correlated with genome duplication and other physiological aspects of the different yeasts. All of our results will be made available to the scientific community using a web accessible database. These studies are expected to help us understand how eukaryotic regulatory networks operate and evolve.